Nitrilases

Abstract

The invention relates to a nitrilase having improved activity in the reaction of a nitrile to form the corresponding carboxylic acid, in particular with respect to reacting 2-methylglutaronitrile, 1-(cyanomethyl)cyclohexane-1-carbonitrile, and benzonitrile. The nitrilase according to the invention is related to nitrilase from acidovorax facilis.

The invention claimed is: 
     
       1. A nitrilase comprising an amino acid sequence having at least 85% homology with the amino acid sequence of SEQ ID NO: 2, which, in comparison with the nitrilase having the amino acid sequence of SEQ ID NO: 2,
 (i) has an activity increased by at least 15% in the reaction of a nitrile to the corresponding carboxylic acid, wherein the nitrile is selected from the group consisting of 2-methylglutaronitrile, 1-(cyanomethyl)cyclohexane-1-carbonitrile and benzonitrile;
 and/or 
 
 (ii) has at least one amino acid substitution selected from the group consisting of:
 a) substitution of Leu in the position which corresponds to position 9 of SEQ ID NO: 2 by an amino acid selected from the group consisting of Arg, Glu, Ile, and Lys; 
 b) substitution of Val in the position which corresponds to position 63 of SEQ ID NO: 2 by an amino acid selected from the group consisting of Ile, Met, Pro, and Thr; 
 c) substitution of Thr in the position which corresponds to position 70 of SEQ ID NO: 2 by an amino acid selected from the group consisting of Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, Ile, Leu, Met, Phe, Pro, Ser, Tyr and Val; 
 d) substitution of Arg in the position which corresponds to position 94 of SEQ ID NO: 2 by an amino acid selected from the group consisting of Ala, Asn, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Tyr and Val; 
 e) substitution of Leu in the position which corresponds to position 194 of SEQ ID NO: 2 by an amino acid selected from the group consisting of Cys, Lys, Met, and Tyr; 
 f) substitution of Thr in the position which corresponds to position 208 of SEQ ID NO: 2 by an amino acid selected from the group consisting of Ala, Gly, and Leu; 
 g) substitution of Cys in the position which corresponds to position 250 of SEQ ID NO: 2 by an amino acid selected from the group consisting of Ala, Arg, Gly, His, Lys, Met, Phe, Ser, Thr, and Tyr; 
 h) substitution of Val in the position which corresponds to position 305 of SEQ ID NO: 2 by an amino acid selected from the group consisting of Leu and Met; and 
 i) substitution of Asp in the position which corresponds to position 308 of SEQ ID NO: 2 by an amino acid selected from the group consisting of Asn, and Trp. 
 
 
     
     
       2. The nitrilase as claimed in  claim 1 , which has at least two amino acid substitutions selected from the group consisting of a), b), c), d), e), f), g), h) and i). 
     
     
       3. The nitrilase as claimed in  claim 1 , which has at least three amino acid substitutions selected from the group consisting of a), b), c), d), e), f), g), h) and i). 
     
     
       4. The nitrilase as claimed in  claim 1 , which additionally has at least one further amino acid substitution selected from the group consisting of:
 j) substitution of Tyr in the position which corresponds to position 65 of SEQ ID NO: 2 by an amino acid selected from the group consisting of Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp and Val; 
 k) substitution of Phe in the position which corresponds to position 168 of SEQ ID NO: 2 by an amino acid selected from the group consisting of Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Pro, Ser, Thr, Trp, Tyr and Val; 
 l) substitution of Phe in the position which corresponds to position 174 of SEQ ID NO: 2 by an amino acid selected from the group consisting of Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Pro, Ser, Thr, Trp, Tyr and Val; 
 m) substitution of Ile in the position which corresponds to position 201 of SEQ ID NO: 2 by an amino acid selected from the group consisting of Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr and Val; and 
 n) substitution of Thr in the position which corresponds to position 210 of SEQ ID NO: 2 by an amino acid selected from the group consisting of Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Tip, Tyr and Val. 
 
     
     
       5. The nitrilase as claimed in  claim 1 , having a residual activity after a temperature treatment that is at least 10% more than the residual activity of a nitrilase having the sequence of SEQ ID NO: 2 after the temperature treatment, wherein the residual activity is with respect to the reaction of a nitrile to the corresponding carboxylic acid, wherein the nitrile is selected from the group consisting of 2-methylglutaronitrile, 1-(cyanomethyl)cyclohexane-1-carbonitrile and benzonitrile and wherein the temperature treatment is incubating the nitrilase in 100 mm potassium phosphate buffer pH 7.0 with 1 mm DTT for 15 min at 55° C. 
     
     
       6. A nitrilase comprising an amino acid sequence having at least 98% homology with the amino acid sequence of SEQ ID NO: 8. 
     
     
       7. A method for producing a carboxylic acid from nitriles by contacting a nitrile with a nitrilase as claimed in  claim 1 . 
     
     
       8. The method as claimed in  claim 7  for producing a carboxylic acid selected from the group consisting of 4-cyanopentanoic acid, 3-hydroxyvaleric acid and glycolic acid. 
     
     
       9. A nitrilase of  claim 1  comprising the amino acid sequence of SEQ ID NO: 2 having one or more amino acid substitution and having an activity that is increased by at least 15% in the reaction of a nitrile to the corresponding carboxylic acid, wherein the nitrile is selected from the group consisting of 2-methylglutaronitrile, 1-(cyanomethyl)cyclohexane-1-carbonitrile and benzonitrile, wherein the at least one amino acid substitutions is selected from the group consisting of:
 a) substitution of Leu in the position which corresponds to position 9 of SEQ ID NO: 2 by Lys, Glu, Ile or Arg; 
 b) substitution of Val in the position which corresponds to position 63 of SEQ ID NO: 2 by Pro, Ile, Met or Thr; 
 c) substitution of Thr in the position which corresponds to position 70 of SEQ ID NO: 2 by Ile, Ala, Cys, Asp, Glu, Phe, Gly, Leu, Met, Asn, Pro, Gln, Ser, Tyr; Arg or Val; 
 d) substitution of Arg in the position which corresponds to position 94 of SEQ ID NO: 2 by Gln, Glu, Phe, Gly, Lys, Met, Asn, Pro, Ala, Gly, His, Ile, Leu, Ser, Thr, Val or Tyr; 
 e) substitution of Leu in the position which corresponds to position 194 of SEQ ID NO: 2 by Tyr, Cys, Lys or Met; 
 f) substitution of Thr in the position which corresponds to position 208 of SEQ ID NO: 2 by Ala, Gly or Leu; 
 g) substitution of Cys in the position which corresponds to position 250 of SEQ ID NO: 2 by Gly, Ala, Phe, Lys, Met, Arg, Ser, Thr, Tyr or His; 
 h) substitution of Val in the position which corresponds to position 305 of SEQ ID NO: 2 by Leu or Met; and 
 i) substitution of Asp in the position which corresponds to position 308 of SEQ ID NO: 2 by Asn or Trp. 
 
     
     
       10. A nitrilase of  claim 4  comprising the amino acid sequence of SEQ ID NO: 2 wherein at least one of the amino acid substitutions is selected from the group consisting of:
 a) substitution of Leu in the position which corresponds to position 9 of SEQ ID NO: 2 by Lys, Glu, Ile or Arg; 
 b) substitution of Val in the position which corresponds to position 63 of SEQ ID NO: 2 by Pro, Ile, Met or Thr; 
 c) substitution of Thr in the position which corresponds to position 70 of SEQ ID NO: 2 by Ile, Ala, Cys, Asp, Glu, Phe, Gly, Leu, Met, Asn, Pro, Gln, Ser, Tyr; Arg or Val; 
 d) substitution of Arg in the position which corresponds to position 94 of SEQ ID NO: 2 by Gln, Glu, Phe, Gly, Lys, Met, Asn, Pro, Ala, Gly, His, Ile, Leu, Ser, Thr, Valor Tyr; 
 e) substitution of Leu in the position which corresponds to position 194 of SEQ ID NO: 2 by Tyr, Cys, Lys or Met; 
 f) substitution of Thr in the position which corresponds to position 208 of SEQ ID NO: 2 by Ala, Gly or Leu; 
 g) substitution of Cys in the position which corresponds to position 250 of SEQ ID NO: 2 by Gly, Ala, Phe, Lys, Met, Arg, Ser, Thr, Tyr or His; 
 h) substitution of Val in the position which corresponds to position 305 of SEQ ID NO: 2 by Leu or Met; 
 i) substitution of Asp in the position which corresponds to position 308 of SEQ ID NO: 2 by Asn or Trp 
 
       and wherein at least one amino acid substitution is selected from the group consisting of
 j) substitution of Tyr in the position which corresponds to position 65 of SEQ ID NO: 2 by an amino acid selected from the group consisting of Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp and Val; 
 k) substitution of Phe in the position which corresponds to position 168 of SEQ ID NO: 2 by an amino acid selected from the group consisting of Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Pro, Ser, Thr, Trp, Tyr and Val; 
 j) substitution of Phe in the position which corresponds to position 174 of SEQ ID NO: 2 by an amino acid selected from the group consisting of Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Pro, Ser, Thr, Trp, Tyr and Val; 
 m) substitution of Ile in the position which corresponds to position 201 of SEQ ID NO: 2 by an amino acid selected from the group consisting of Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr and Val; and 
 n) substitution of Thr in the position which corresponds to position 210 of SEQ ID NO: 2 by an amino acid selected from the group consisting of Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Trp, Tyr and Val. 
 
     
     
       11. The nitrilase as claimed in  claim 9 , which has at least two amino acid substitutions selected from the group consisting of a), b), c), d), e), f), g), h) and i). 
     
     
       12. The nitrilase as claimed in  claim 9 , which has at least three amino acid substitutions selected from the group consisting of a), b), c), d), e), f), g), h) and i). 
     
     
       13. The nitrilase as claimed in  claim 9 , having a residual activity after a temperature treatment that is increased by at least 10% with respect to the reaction of a nitrile to the corresponding carboxylic acid, wherein the nitrile is selected from the group consisting of 2-methylglutaronitrile, 1-(cyanomethyl)cyclohexane-carbonitrile and benzonitrile wherein the temperature treatment is incubating the nitrilase in 100 mM potassium phosphate buffer pH 7.0 with 1 mM DTT for 15 min at 55° C. or 60° C. 
     
     
       14. A method for producing a carboxylic acid from nitriles by contacting a nitrile with a nitrilase as claimed in  claim 9 . 
     
     
       15. The method as claimed in  claim 14  for producing a carboxylic acid selected from the group consisting of 4-cyanopentanoic acid, 3-hydroxyvaleric acid and glycolic acid. 
     
     
       16. A nitrilase comprising an amino acid sequence having at least 85% homology with the amino acid sequence of SEQ ID NO: 2, which, in comparison with the nitrilase having the amino acid sequence of SEQ ID NO: 2,
 (i) has an activity increased by at least 15% in the reaction of a nitrile to the corresponding carboxylic acid, wherein the nitrile is selected from the group consisting of 2-methyl glutaronitrile, 1-(cyanomethyl)cyclohex ane-1-carbonitrile and benzonitrile; and 
 (ii) has at least one amino acid substitution selected from the group consisting of:
 a) substitution of Leu in the position which corresponds to position 9 of SEQ ID NO: 2 by an amino acid selected from the group consisting of Arg, Glu, Ile, and Lys; 
 b) substitution of Val in the position which corresponds to position 63 of SEQ ID NO: 2 by an amino acid selected from the group consisting of Ile, Met, Pro, and Thr; 
 c) substitution of Thr in the position which corresponds to position 70 of SEQ ID NO: 2 by an amino acid selected from the group consisting of Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, Ile, Leu, Met, Phe, Pro, Ser, Tyr and Val; 
 d) substitution of Arg in the position which corresponds to position 94 of SEQ ID NO: 2 by an amino acid selected from the group consisting of Ala, Asn, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Tyr and Val; 
 e) substitution of Leu in the position which corresponds to position 194 of SEQ ID NO: 2 by an amino acid selected from the group consisting of Cys, Lys, Met, and Tyr; 
 f) substitution of Thr in the position which corresponds to position 208 of SEQ ID NO: 2 by an amino acid selected from the group consisting of Ala, Gly, and Leu; 
 g) substitution of Cys in the position which corresponds to position 250 of SEQ ID NO: 2 by an amino acid selected from the group consisting of Ala, Arg, Gly, His, Lys, Met, Phe, Ser, Thr, and Tyr; 
 h) substitution of Val in the position which corresponds to position 305 of SEQ ID NO: 2 by an amino acid selected from the group consisting of Leu, and Met; and 
 i) substitution of Asp in the position which corresponds to position 308 of SEQ ID NO: 2 by an amino acid selected from the group consisting of Asn and Tip.